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WO2004055117A2 - Colorants substitues par carboxamide pour applications analytiques - Google Patents

Colorants substitues par carboxamide pour applications analytiques Download PDF

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Publication number
WO2004055117A2
WO2004055117A2 PCT/EP2003/014534 EP0314534W WO2004055117A2 WO 2004055117 A2 WO2004055117 A2 WO 2004055117A2 EP 0314534 W EP0314534 W EP 0314534W WO 2004055117 A2 WO2004055117 A2 WO 2004055117A2
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Prior art keywords
carboxamide
substituted
group
dye
general formula
Prior art date
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PCT/EP2003/014534
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German (de)
English (en)
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WO2004055117A3 (fr
Inventor
Jutta Arden-Jacob
Karl-Heinz Drexhage
Monika Hamers-Schneider
Norbert Kemnitzer
Alexander Zilles
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ATTO-TEC GmbH
ATTO TEC GmbH
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ATTO-TEC GmbH
ATTO TEC GmbH
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Priority to EP03799491A priority Critical patent/EP1576059B1/fr
Priority to US10/539,790 priority patent/US7935822B2/en
Priority to AU2003300216A priority patent/AU2003300216A1/en
Publication of WO2004055117A2 publication Critical patent/WO2004055117A2/fr
Publication of WO2004055117A3 publication Critical patent/WO2004055117A3/fr
Anticipated expiration legal-status Critical
Priority to US13/051,561 priority patent/US8530660B2/en
Priority to US13/962,736 priority patent/US8785637B2/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • the present invention relates to carboxamide-substituted dyes, their preparation and the use of such dyes as marker groups in analysis.
  • Dyes from the class of xanthene and triphenylmethane dyes and related derivatives are preferably used as labeling groups in chemical, biological and medical analysis due to their very good spectral properties (J. Slavik, Fluorescent Probes in Cellular and Molecular Biology, CRC Press, Boca Raton, Ann Arbor, London, Tokyo, 1994).
  • Dyes from related classes of carbopyronins and amidopyrylium dyes are described in WO 00/64986 and WO 00/64987. Dyes with a very high fluorescence quantum yield play an important role here, since fluorescence enables very sensitive detection of the labeled analyte. But non-fluorescent derivatives are also becoming increasingly important as quenchers in special processes.
  • Lactam dye colorless
  • WO 02/055512 discloses the production of amide derivatives of fluorescein dyes, the carboxylic acid group first being converted to an active ester at higher temperatures and then the active ester being reacted with a secondary amine in an aqueous solvent mixture.
  • the method according to WO 02/055512 is only applicable to fluorescein and its derivatives and cannot be transferred to other classes of dyes.
  • the disclosed process conditions such as temperature and solvent used, are not applicable to compounds containing amino and / or imino groups, such as, for example, rhodamines.
  • the temperatures set during the formation of the active ester lead to by-products and decomposition products; on the other hand, the water used in the reaction of the active ester with an amine as solvent can react with the active ester, which in turn results in undesired products.
  • Y oxygen, sulfur, selenium, CR a R b , NR C , a direct link or -R 14 and -R 15 ;
  • R 3 R 4 independently represent hydrogen, halogen (such as fluorine, chlorine, bromine, iodine), -O ⁇ , a hydroxy group, thiol group, amino group, ammonium group, sulfo group, phospho group, nitro group, carbonyl group (e.g. keto or aldehyde group), carboxy group Carboxylic acid derivative (such as carboxylate, ester, halide, amide, anhydride), nitrile group, isonitrile group, cyanate group, isocyanate group, thiocyanate group, isothiocyanate group or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon radical having up to 40 carbon atoms;
  • R a , R b , R G and R 14 , R 15 independently have the same meanings as defined for R ,, R 3 , R 4 ;
  • R 2 O; g_ Rg or ⁇ ⁇ R?
  • R 7 , R 8 , R 9 independently represent hydrogen or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon radical having up to 40 carbon atoms; or R. together with R 2
  • Rio / Ri v R 13 have the same meanings as defined for R 1 f R 3 , R 4 ;
  • R-ie, R ⁇ 7 , R 18 have the same meanings as it is defined for R 7 , R 8 , R 9 ;
  • R 5 , R 6 independently represent a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon radical having up to 40 carbon atoms;
  • Cyd represents an organic radical comprising a ring system selected from aromatic, heteroaromatic, quinoid and cycloaliphatic rings;
  • Cyc2 represents an organic radical comprising a ring system selected from aromatic, heteroaromatic, quinoid and cycloaliphatic rings;
  • Cyd phenyl or substituted phenyl
  • Cyc2 hydroxy, ether or ester substituted phenyl
  • - R 2 O.
  • Cyd phenyl or substituted phenyl
  • Cyc2 phenyl or substituted phenyl
  • the properties of the dye can also be controlled by introducing various residues on the amide group.
  • the lipophilicity of the dye can be increased by introducing long alkyl chains as residues on the amide group.
  • by introducing e.g. Sugar residues or other polar groups increase the hydrophilicity of the dye. This allows properties such as solubility behavior to be modulated in a simple manner.
  • various linkers can be installed on the amide group in order to e.g. to be able to couple or conjugate to an analyte to be detected, such as a peptide or the like.
  • the hydrocarbon groups include alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aryl groups and heteroaryl groups. These groups can be heteroatoms, such as oxygen, Sulfur or / and nitrogen. Furthermore, further substituents can be attached to these groups, which are preferably selected from halogen (such as fluorine, chlorine, bromine, iodine), -O ⁇ , a hydroxyl group, thiol group, amino group, ammonium group, sulfo group, phospho group, nitro group, carbonyl group (e.g. Keto or aldehyde group), carboxy group, a carboxylic acid derivative (e.g.
  • alkyl groups here includes straight-chain or branched C, -C 40 alkyl groups, preferably C 2 -C 35 alkyl groups, more preferably C 5 -C 30 alkyl groups, even more preferably C 8 -C 20 alkyl groups.
  • the alkyl groups are selected from methyl, ethyl and straight or branched propyl, butyl, hexyl, decyl, dodecyl and octadecyl groups.
  • alkenyl groups include straight-chain or branched C 2 -C 40 alkenyl groups with one or more double bonds at any point in the hydrocarbon radical. Depending on the chain length, 1 -10, 2-8 or 4-6 double bonds per residue are preferred.
  • the alkenyl radicals have 2-40, preferably 4-35, more preferably 8-25, even more preferably 15-20 carbon atoms. Examples of suitable alkenyl groups are ethenyl, propenyl, butenyl, hexenyl, decenyl, dodecenyl and octadecenyl.
  • Hydrocarbon residues Depending on the chain length, 1 -10, 2-8 or 4-6
  • the alkynyl radicals have 2-40, preferably 4-35, more preferably 8-25, even more preferably 15-20 carbon atoms.
  • suitable alkynyl groups are ethynyl, propynyl, butynyl, hexynyl, decynyl, dodecynyl and octadecynyl.
  • the cycloalkyl groups can be saturated or unsaturated, ie optionally have one or more double or / and triple bonds.
  • Preferred are C 3 -C 40 cycloalkyl groups, more preferably C 3 - C 20 cycloalkyl groups, more preferably C 4 -C 12 cycloalkyl groups, more preferably C 5 - or C 6 -cycloalkyl.
  • suitable cycloalkyl groups are selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl and cyclooctyl.
  • C 3 -C 40 cycloalkyl groups can be bridged and form bicyclic or polycyclic compounds.
  • polycyclic residues are norbornane, norbornene and bicyclo [3.2.2] octyl residues or the respective substituted derivatives thereof.
  • the cycloalkyl groups also include aliphatic heterocycles such as tetrahydropyrrole, piperidine, dioxane, tetrahydrofuran groups.
  • Preferred aryl radicals are C 3 -C 40 aryl groups, more preferably C 6 -C 20 aryl groups and C 10 -C 14 aryl groups.
  • suitable aryl groups are phenyl, naphthyl, anthracene and phenanthrene groups.
  • the heteroaryl groups represent aromatic radicals with 3-40 carbon atoms, which have one or more heteroatoms, the heteroatoms being selected from oxygen, sulfur and nitrogen.
  • the heteroaryl groups preferably contain 1-10, preferably 2-6, more preferably 3-5 heteroatoms.
  • the heteroaryl groups contain 3-40, especially 3-18, more preferably 4-14, even more preferably 5-8 carbon atoms.
  • suitable heteroaryl groups are pyrrole, pyridine, pyrimidine, indole, furan, thiophene and thiazole groups.
  • Hydrocarbon groups which contain heteroatoms are, for example, heteroatom-containing alkyl groups, for example straight-chain or branched C
  • suitable alkoxy groups are methoxy, ethoxy, straight-chain or branched propoxy, butoxy, decoxy and undecoxy.
  • heteroatom-containing aryl groups examples include aryloxy groups such as phenoxy and naphthoxy.
  • Heteroatom-containing hydrocarbon residues according to the present invention further include e.g. Carbonyl groups (such as keto or aldehyde groups), carboxylic acids, carboxylic acid derivatives (such as halides, esters, amides, anhydrides, caboxylates), ethers, thioethers and alkoxycarbonyl groups which have 1-40 carbon atoms, preferably 2-35, more preferably 5-30 and also preferably contain 8-20 carbon atoms.
  • Carbonyl groups such as keto or aldehyde groups
  • carboxylic acids such as halides, esters, amides, anhydrides, caboxylates
  • ethers, thioethers such as halides, esters, amides, anhydrides, caboxylates
  • alkoxycarbonyl groups which have 1-40 carbon atoms, preferably 2-35, more preferably 5-30 and also preferably contain 8-20 carbon atoms.
  • Cyd and Cyc2 can also be further substituted, the substituents preferably being selected from halogen (such as fluorine, chlorine, bromine, iodine), -O ⁇ , a hydroxyl group, thiol group, amino group, ammonium group, sulfo group, phospho group, nitro group, carbonyl group (e.g.
  • each of the radicals contained in the carboxamide-substituted dye of the general formula (I) according to the invention can optionally form a ring system with one or more adjacent radicals.
  • the ring system preferably contains 5- or / and 6-membered rings.
  • the anions optionally required for charge balance can be selected from inorganic and / or organic anions, such as e.g. Halides, sulfates, carbonates, phosphates, sulfites, sulfides, hydroxides, alkoxides, carboxylates, nitrates, nitrites etc.
  • inorganic and / or organic anions such as e.g. Halides, sulfates, carbonates, phosphates, sulfites, sulfides, hydroxides, alkoxides, carboxylates, nitrates, nitrites etc.
  • the carboxamide-substituted dyes of the general formula (I) according to the invention are preferably dyes in which Cyc2 in formula (I) represents a nitrogen-containing heterocycle or a ring system which is substituted by at least one amino group. It is further preferred that R 2
  • R 12 means.
  • Cyc2 in formula (I) has one of the structures (A), (B), (C), (D), (E), (F), (G) (H) or (J ) on:
  • R 19 , R 20 and R 22 , R 23 are independently defined as R 7 , R 8 ;
  • R 21 is defined as R 7 and the dashed lines if necessary
  • Double bonds mean, in the presence of which the residues bound by a dashed line are missing.
  • the substructures (A) to (J) are condensed onto the remaining ring structure of the formula (I) in such a way that the
  • Linking points are connected to one another by single or double bonds. Preferably lie between the linking points
  • adjacent substituents can optionally form further ring systems as defined above.
  • the radicals R 19 , R 20 and R 22 , R 23 with the adjacent radicals R preferably form ring systems which comprise 5- or / and 6-membered rings which may optionally contain further heteroatoms. It is particularly preferred that Cyc2 has one of the structures (A), (D), (E), (F), (G), (H) or (J).
  • the ring systems formed from R 7 or R 8 with adjacent substituents preferably lead to the following systems (K), (L), (M), (N) or (O):
  • R is each independently defined as R ,, R 3 , R 4 , and the dashed lines optionally signify double bonds, in the presence of which the residues bound by a dashed line are missing.
  • neighboring substituents can also form further ring systems as defined above.
  • Cyd represents an optionally substituted organic radical which comprises a ring system which is preferably selected from substituted and unsubstituted phenyl, naphthyl, pyridyl and cyclohexyl.
  • the radicals R 5 or / and R 6 of the carboxamide group preferably have a substituent which is suitable for coupling to other molecules.
  • a covalent coupling can particularly preferably be achieved via such a substituent.
  • Suitable substituents on R 5 and / or R 6 are a carboxyl group, amino group, hydroxyl group, thiol group, cyano group, halogen group and / or groups with unsaturated units, such as double and triple bonds.
  • the dyes according to the invention can be bound, for example, to biomolecules or carriers.
  • At least one of the radicals R 5 and R 6 represents a carboxy-substituted alkyl group, the radical not substituted by a carboxyl group preferably representing an alkyl group.
  • the properties of the resulting dye molecules with regard to solubility and fluorescence properties can also be adjusted and modified in a simple manner. If R 5 or / and R 6 carry, for example, longer alkyl chains (eg Tab. 2, Verb. 56 and 57), the lipophilic character increases, and the compound is soluble in non-polar media and membranes and can thus be used, for example, for the detection of membrane properties or be used for molecular distance measurements.
  • R 5 or / and R 6 carry, for example, longer alkyl chains (eg Tab. 2, Verb. 56 and 57)
  • the lipophilic character increases, and the compound is soluble in non-polar media and membranes and can thus be used, for example, for the detection of membrane properties or be used for molecular distance measurements.
  • the water solubility of a dye can be improved, for example, if R 5 or / and R 6 carry, for example, sulfonic or phosphonic acid groups (for example Tab. 2, Verb. 55) or have polyether chains. The latter also make the compound more soluble in many organic solvents.
  • the crown ethers used for fluorescence-sensitive cation detection represent a kind of special polyether, which as aza derivatives can also be coupled to dye molecules using the carboxamide method (eg Tab. 2, Verb. 53).
  • sugar residues can be introduced as R 5 and / or R 6 , whereby a high water solubility can be achieved (see, for example, Tab. 2, Verb. 51 NK67).
  • the dyes according to the invention also have the advantage that the properties of the carboxamide-substituted dyes can be varied depending on the intended use by different meanings of Y.
  • Such connections include valuable as a fluorescence quencher in bioanalytics.
  • Examples of such carboxamide-substituted triphenylmethane dyes are the compounds 29 and 30 according to the invention in Table 1 (NK88, NK 89).
  • Another aspect of the present invention is a multichromophoric system in which a carboxamide-substituted dye according to the general formula (I) is coupled to one or more further dye molecules via R 5 and / or R 6 .
  • the multichromophore system according to the invention is preferably a system in which a further dye molecule according to the general formula (I) is coupled to a carboxamide-substituted dye according to the invention.
  • a bichromophoric system is compound 59 in Table 3 (NK69).
  • the coupling to the further carboxamide-substituted dyes is carried out via the radical R 5 or / and R 6 .
  • the nitrogen atoms of the carboxamide group in the dye units are linked via alkyl chains.
  • Another preferred embodiment of the multichromophoric system according to the invention is a bichromophoric system of the formula (III).
  • R 24 , R 25 and R 26 , R 27 are defined as R 7 , R 8 , where n is independently 0, 1, 2 or 3 and m is 0, 1, 2, 3 or 4 ,
  • FRET energy transfer
  • the carboxamide-substituted dyes according to the invention are distinguished in particular by the fact that they do not show the lactone or lactam formation which occurs with conventional carboxyl-substituted dyes.
  • the carboxamide group By introducing the carboxamide group, it can also be achieved that the dye molecules can be functionalized in a variable manner and are therefore excellently suitable for a wide variety of applications, such as analytical processes.
  • the introduction of the carboxamide group does not significantly change the very good spectral properties of the starting dyes, that is to say the absorption and fluorescence bands, the high extinction coefficients and high fluorescence quantum yields and stability. With some dyes it was only observed that the Absorption and fluorescence maximum of the dyes is shifted by an average of 10 nm long-wave.
  • the present invention therefore furthermore relates to a process for the preparation of carboxamide-substituted dyes of the general formula (I), comprising the steps:
  • step (b) reacting the activated dye obtained in step (a) with a secondary amine HNR 5 R 6 ; and (c) optionally isolating the carboxamide-substituted dye of the general formula (I) obtained in step (b).
  • the activation of a carboxyl-containing dye according to general formula (II) is preferably carried out by converting the carboxyl group into an active ester or an acid chloride. Activation is carried out using standard methods well known to those skilled in the art. In general, reagents commonly used to prepare the active esters, e.g. N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxynaphthalimide, O- (N-succinimidyl) -N, N, N ', N'-tetramethyluronimtetrafluoroborate (TSTU) are used. The process is preferably carried out at from 0 ° C. to about 60 ° C., more preferably at 10 ° C. to 40 ° C., most preferably at 20 ° C. to 30 ° C., in particular at room temperature.
  • TSTU tetramethyluronimtetrafluoroborate
  • the production of activated dyes derived from rhodamine is carried out at room temperature in order to avoid decomposition of the rhodamine derivatives.
  • the reaction times vary depending on the structure of the dye, but in general the reaction is complete after about 5-48 hours, preferably 8-24 hours.
  • the activated dye obtained in step (a) is reacted with secondary amines which contain the radicals R 5 and R 6 desired later in the carboxamide-substituted dye of the formula (I).
  • the reaction is preferably carried out at room temperature up to temperatures of approximately 100 ° C, preferably at temperatures of approximately 50 ° C to 90 ° C, more preferably approximately 60 ° C to 80 ° C.
  • the active ester and the corresponding secondary amine are combined in an aprotic one Solvents such as acetonitrile, DMF, DMSO etc., dissolved and heated (carboxamide process).
  • care must be taken to ensure that the solvent is anhydrous in order to avoid a reaction with the active ester.
  • the carboxamide substituted dye obtained in step (b) is obtained by methods well known to those skilled in the art, such as e.g. Extracting, filtering, working up by column chromatography, distillation, etc., isolated from the reaction mixture.
  • a major advantage of the process described is the unrestricted applicability to all dyes of the general formula (II) which, in particular, have a carboxyl group which is in the ortho position or which tends to form lactones due to their position.
  • Examples of dye classes that can be used are shown in FIG. 1. It is particularly noteworthy that the process according to the invention also contains dyes containing amino and imino or iminium groups, such as, for example, Rhodamine, can be successfully implemented.
  • the resulting dyes according to the invention are particularly suitable as labeling groups in analytical processes.
  • Another object of the present invention is therefore the use of a carboxamide dye according to the invention according to the general formula (I) for the qualitative and / or quantitative determination of an analyte.
  • the dyes according to the invention show very good results in chemical as well as in medical and biological detection methods.
  • the determination can be made, for example, in aqueous liquids such as body fluids, for example blood, serum, plasma or urine, Waste water samples or food can be carried out using detection methods known to those skilled in the art.
  • the method can be carried out both as a wet test, for example in a cuvette, or as a dry test on an appropriate reagent carrier.
  • the analytes can be determined using a single reaction or a sequence of reactions.
  • the carboxamide-substituted dye of the invention is preferably coupled to the analyte or / and a support.
  • the dye is coupled to a component of a detection reagent.
  • a component is preferably a universal receptor or an analyte analog.
  • binding partners are preferably selected from peptides, polypeptides, nucleic acids, nucleosides, nucleotides, nucleic acid analogs and haptens.
  • the detection includes in particular an immunological detection and / or a nucleic acid hybridization detection.
  • the analyte to be detected is preferably selected from peptides, polypeptides, antibodies, nucleic acids, nucleic acid analogs, haptens, cells, cell components, viruses, viral components, metabolites, hormones, neurotransmitters and drugs.
  • any suitable material can be selected as the carrier, e.g. porous glass, plastics, ion exchange resins, dextrans, cellulose, cellulose derivatives and hydrophilic polymers.
  • Another object of the present invention is a conjugate of a binding partner with a carboxamide-substituted dye according to the formula
  • the binding partner is preferably selected from peptides,
  • conjugates can be used, for example, in nucleic acid hybridization processes or immunochemical processes. Such methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor.
  • the coupling to the analyte to be detected or / and the constituent of a detection reagent or / and the carrier is preferably carried out via the substituents R 5 or / and R 6 of the carboxamide-substituted dye of the general formula (I).
  • a covalent bond is particularly preferably formed. In particular, this is accomplished using the active ester method, which is well known to those skilled in the art.
  • Carboxyl groups or substituents with carboxyl groups as radicals R 5 or R 6 are particularly suitable for this.
  • Such a carboxyl group can in turn be activated and can preferably be used for coupling and conjugate formation of the dye according to the invention to analytes, constituents of detection reagents or carriers.
  • succinimidyl ester prepared from the dye NK 50 (compound 1) completely converts to the corresponding conjugate within 30 minutes using benzylamine in acetonitrile according to HPLC or DC analysis. Even with aminoethyl maleimide, complete conversion to the maleimide derivative is obtained after a few hours. This compound was successfully reacted with N-acetylcysteine. With the described method, all common derivatives can be produced from the terminal carboxyl group, e.g. can be used for coupling to amino and thiol groups of biomolecules.
  • the carboxamide-substituted dyes according to the invention have the advantage that they do have an activatable carboxyl group, but this does not tend to lactone or lactam formation.
  • FIG. 2 shows carboxamide-substituted dyes according to the invention with a carboxyl group which can be coupled to R 5 or R 6 and the respective spectral data (absorption maximum, fluorescence maximum and fluorescence quantum yield).
  • FIG. 3 shows further examples of carboxamide-substituted dyes according to the invention and their spectral data (absorption maximum, fluorescence maximum and fluorescence quantum yield).
  • FIG. 4 shows bichromophoric systems according to the invention.
  • Figure 5 shows the absorption or fluorescence spectrum of compound NK51 in ethanol.
  • Figure 6 shows the absorption or fluorescence spectrum of compound NK56 in ethanol.
  • Figure 7 shows the absorption or fluorescence spectrum of compound NK63 in ethanol.
  • Figure 8 shows the absorption or fluorescence spectrum of compound NK65 in ethanol.
  • the dyes used for the carboxamide process are either commercially available or can be prepared using syntheses known from the literature or processes known to the person skilled in the art. In most cases, the secondary amines used are also commercially available or can be obtained by methods known from the literature. The preferred method for producing compounds according to the invention is described by way of example using structures 1 (NK 50), 4 (NK 56), 51 (NK 67) and 1 8 (NK 79).
  • the active ester is precipitated by rapid dropwise addition of ice-cold 10% sodium perchlorate solution, suction filtered, washed with ice water and diethyl ether and finally dried in a vacuum desiccator over phosphorus pentoxide. Yield: 0.8 g
  • the raw product can be used for the next stage without cleaning.
  • the product can be dissolved in dilute sodium hydroxide solution and reprecipitated with half-concentrated sulfuric acid.
  • NK 135 100 mg (0.3 mmol) of NK 135 are dissolved in 10 ml of acetonitrile with the addition of approximately 1 00 ⁇ ⁇ DM F and 0.5 ml of 20% aqueous tetraethylammonium hydroxide solution and with 72 mg (0.35 mmol) of dicyclohexylcarbodiimide and 40 mg (0.35 mmol) N-hydroxysuccinimide added.
  • the solution is stirred for 12 hours at room temperature, then half concentrated in vacuo at about 40 ° C., filtered and then a solid is precipitated by adding water.
  • the crude product is filtered off, washed with a little water and dried in vacuo over phosphorus pentoxide. The solid also contains unreacted starting material.
  • NK 50 perchlorate 0.5 g (0.78 mmol) of NK 50 perchlorate are dissolved in 20 ml of dry acetonitrile, 250 mg (0.85 mmol) of TSTU and 170 l (1 mmol) of Hunig base are added and the mixture is stirred at room temperature.
  • the solution is concentrated in vacuo to about a quarter, 1 ml of 60% perchloric acid is added and the dye is precipitated by the dropwise addition of 20% sodium perchlorate solution.
  • NK 50-NHS esters 0.1 mmol NK 50-NHS esters are dissolved in 5 ml dry acetonitrile, 0.15 mmol aminoethyl maleinimide and 30 ⁇ ⁇ Hunig base are added. The solution is stirred for 5 hours at room temperature, filtered and dropped in diethyl ether. The precipitate thus obtained is dried in an oil pump vacuum.
  • NK 50-aminoethyl-maleinimide 0.1 mmol are dissolved in 20 ml of ethanol and a total of 0.1 5 mmol of cysteine are added in portions. The mixture is stirred at room temperature and about 50 ml of a 10% sodium perchlorate solution are added dropwise after about 2 hours. The precipitated solid is filtered off and dried in a vacuum desiccator over phosphorus pentoxide.
  • NK 50-digoxin-3-carboxymethyl ether-diaminodioxaoctane conjugate (Dig-CME-DADOO)
  • NK 50 active ester 0.02 mmol NK 50 active ester are stirred with 0.02 mmol Dig-CME-DADOO in acetonitrile for 18 hours at room temperature. The solution is concentrated and the residue is purified by chromatography.

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  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne des colorants substitués par carboxamide, ainsi que leur production et leur utilisation comme groupes de marquage en analytique.
PCT/EP2003/014534 2002-12-18 2003-12-18 Colorants substitues par carboxamide pour applications analytiques Ceased WO2004055117A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP03799491A EP1576059B1 (fr) 2002-12-18 2003-12-18 Colorants substitues par carboxamide pour applications analytiques
US10/539,790 US7935822B2 (en) 2002-12-18 2003-12-18 Carboxamide-substituted dyes for analytical applications
AU2003300216A AU2003300216A1 (en) 2002-12-18 2003-12-18 Carboxamide-substituted dyes for analytical applications
US13/051,561 US8530660B2 (en) 2002-12-18 2011-03-18 Carboxamide-substituted dyes for analytical applications
US13/962,736 US8785637B2 (en) 2002-12-18 2013-08-08 Carboxamide-substituted dyes for analytical applications

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10259374A DE10259374A1 (de) 2002-12-18 2002-12-18 Carboxamid-substituierte Farbstoffe für analytische Anwendungen
DE10259374.4 2002-12-18

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US13/051,561 Division US8530660B2 (en) 2002-12-18 2011-03-18 Carboxamide-substituted dyes for analytical applications

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US (3) US7935822B2 (fr)
EP (1) EP1576059B1 (fr)
AU (1) AU2003300216A1 (fr)
DE (1) DE10259374A1 (fr)
WO (1) WO2004055117A2 (fr)

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CN113321638A (zh) * 2021-06-09 2021-08-31 南京溯远基因科技有限公司 适用于各种荧光检测场景的高性能荧光染料及制备方法

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US9228225B2 (en) 2004-02-03 2016-01-05 Biosearch Technologies, Inc. Xanthene dyes
US8178360B2 (en) 2006-05-18 2012-05-15 Illumina Cambridge Limited Dye compounds and the use of their labelled conjugates
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CN113321638A (zh) * 2021-06-09 2021-08-31 南京溯远基因科技有限公司 适用于各种荧光检测场景的高性能荧光染料及制备方法

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US8530660B2 (en) 2013-09-10
US20130323851A1 (en) 2013-12-05
US20060154251A1 (en) 2006-07-13
EP1576059A2 (fr) 2005-09-21
US8785637B2 (en) 2014-07-22
WO2004055117A3 (fr) 2004-08-19
AU2003300216A1 (en) 2004-07-09
EP1576059B1 (fr) 2012-06-27
AU2003300216A8 (en) 2004-07-09
US20110223677A1 (en) 2011-09-15
DE10259374A1 (de) 2004-07-08
US7935822B2 (en) 2011-05-03

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